Plant and Cell Physiology

The circadian clock regulates temporal metabolic activities, enabling organisms to adapt to cyclic environmental changes, but how it affects lipid metabolism in plants is poorly understood. Our previous finding showed that the central clock transcription factors LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) increased seed oil contents in Arabidopsis. Here we investigated the molecular and metabolic mechanism underlying the LHY and CCA1 regulated oil accumulation. Triacylglycerol (TAG) accumulation in Arabidopsis developing seeds was increased in LHY-overexpressing (LHY-OE) and decreased in lhycca1 plants compared to wild-type (WT). Metabolic tracking of lipids in developing seeds indicated that fatty acids (FAs) of major lipid precursors for TAG production increased more rapidly in LHY-OE and slowly in lhycca1 than in WT, suggesting that LHY enhanced FA synthesis. Transcript analysis revealed that the expression of genes involved in FA synthesis, including the one encoding {beta}-ketoacyl-ACP synthase III (KASIII), was oppositely changed in developing seeds of LHY/CCA1-OEs and those of lhycca1. Chromatin immunoprecipitation, electrophoretic mobility shift, and transactivation assays indicated that LHY directly bound and activated the promoter of KASIII. Furthermore, phosphatidic acid, a metabolic precursor to TAG, inhibited LHY binding to KASIII promoter elements. Our data reveal a new regulatory mechanism by the core clock regulators for storage lipid production during plant seed development.

Low-light stress compromises photosynthetic and energy efficiency and leads to spikelet sterility; however, the effect of low-light stress on pollen tube elongation in the pistil remains poorly understood. The gene RGA1, which encodes a G subunit of the heterotrimeric G protein, enhanced low-light tolerance in rice plants at anthesis by preventing the cessation of pollen tube elongation in the pistil. The levels of reactive oxygen species were higher and the content of ATP and ATPase was lower in RGA1 mutant (d1) plants compared with wild-type and RGA1-overexpressing (OE-1) plants under low-light conditions. Energy deficits, rather than interference with signaling transduction pathways, were the main contributors to the inhibition of pollen tube elongation in the pistil by low-light stress. In this process, marked increases in the activities of acid invertase (INV), sucrose synthase (SUS), and mitochondrial respiratory electron transport chain complexes, as well as the relative expression levels of SUTs, SWEETs, SUSs, INVs, CINs, SnRK1A, and SnRk1B, were observed in OE-1 plants. INV and ATPase activators (sucrose and Na2SO3, respectively) increased spikelet fertility by improving the energy status in the pistil under low-light conditions, and the ATPase inhibitor Na2VO4 induced spikelet sterility and decreased ATPase activity. Therefore, RGA1 could alleviate the low-light stress-induced impairment of pollen tube elongation to increase spikelet fertility by promoting sucrose unloading in the pistil and improving the metabolism and allocation of energy.

Plant phospholipase C (PLC) proteins are phospholipid-degrading enzymes classified into two subfamilies: phosphoinositide-specific PLCs (PI-PLCs) and non-specific PLCs (NPCs). PI-PLCs have been widely studied in various biological contexts, including responses to abiotic and biotic stresses and plant development; NPCs have been less thoroughly studied. No PLC subfamily has been characterized in relation to the symbiotic interaction between Fabaceae (legume) species and the nitrogen-fixing bacteria called rhizobia. However, lipids are reported to be crucial to this interaction, and PLCs may therefore contribute to regulating legume-rhizobia symbiosis. In this work, we functionally characterized NPC4 from common bean (Phaseolus vulgaris L.) during rhizobial symbiosis, finding evidence that NPC4 plays an important role in bean root development. The knockdown of PvNPC4 by RNA interference (RNAi) resulted in fewer and shorter primary roots and fewer lateral roots than were seen in control plants. Importantly, this phenotype seems to be related to altered auxin signaling. In the bean-rhizobia symbiosis, PvNPC4 transcript abundance increased 3 days after inoculation with Rhizobium tropici. Moreover, the number of infection threads and nodules, as well as the transcript abundance of PvEnod40, a regulatory gene of early stages of symbiosis, decreased in PvNPC4-RNAi roots. Additionally, transcript abundance of genes involved in autoregulation of nodulation (AON) was altered by PvNPC4 silencing. These results indicate that PvNPC4 is a key regulator of root and nodule development, underscoring the participation of PLC in rhizobial symbiosis.

To discover new mutant alleles conferring enhanced tolerance to drought stress, we screened a mutagenized rice population (cv. IAPAR9) and identified a mutant, named idr1-1 (for increased drought resistance 1-1), with obviously increased drought tolerance under upland field conditions. The idr1-1 mutant possessed a significantly enhanced ability to tolerate high-drought stress in different trials. Map-based cloning revealed that the gene LOC_Os05g26890 (corresponding to D1 or RGA1 gene), residing in the mapping region of IDR1 locus, carried a single-base deletion in the idr1-1 mutant, which caused a frameshift and premature translation termination. Complementation tests indicated that such a mutation was indeed responsible for the elevated drought tolerance in idr1-1 mutant. IDR1 protein was localized in nucleus and to plasma membrane or cell periphery. Further investigations indicated that the significantly increased drought tolerance in idr1-1 mutant stemmed from a range of physiological and morphological changes occurring in such a mutant, including greater leaf potentials, increased proline contents, heightened leaf thickness, and upregulation of antioxidant-synthesizing and drought-induced genes, etc., under drought-stressed conditions. Especially, ROS production from NADPH oxidases and chloroplasts might be remarkably impaired, while ROS-scavenging ability appeared to be markedly enhanced as a result of significantly elevated expression of a dozen ROS-scavenging enzyme genes in idr1-1 mutant under drought-stressed conditions. Besides, IDR1 physically interacted with TUD1, and idr1-1 mutant showed impaired EBR responsiveness. Altogether, these results suggest that mutation of IDR1 leads to alterations of multiple layers of regulations, which ultimately confers obviously enhanced drought tolerance to the idr1-1 mutant. One-sentence summaryMutation of IDR1 significantly enhances drought tolerance in an upland cultivar IAPAR9 by decreasing apoplastic and chloroplastic ROS production and increasing ROS-scavenging ability

Biogenesis and maintenance of the photosynthetic thylakoid membrane requires transport of lipids from their site of synthesis in the chloroplast envelopes to their destination in the thylakoid. While vesicle trafficking is likely involved, we hypothesized a complementary mechanism involving direct membrane interactions. Using domain homology and proteomic profiling of chloroplast membrane fractions, we identified candidate lipid transport proteins enriched in a distinct, intermediate-density membrane population. This fraction was enriched in lipid metabolic enzymes and proteins homologous to known membrane organization factors. Several candidates, including TVP38 FAMILY PROTEIN (TVPFP), PLASMA MEMBRANE FUSION PROTEIN (PMFP), and LETM1-LIKE, localized to discrete subdomains within chloroplasts. Loss- of-function tvpfp or pmfp mutants exhibited altered chloroplast ultrastructure, including changes in thylakoid-envelope proximity, supporting their roles in maintaining membrane architecture. These findings identify a specialized chloroplast membrane region enriched in lipid-related functions and offer a foundation for elucidating the molecular architecture of these regions.

Phosphatidic acid (PA) is considered as a second messenger that interacts with protein kinases, phosphatases and NADPH oxidases, amplifying the signal to initiate plant defense signaling responses (Li and Wang, 2019). In rice, mutation of RBL1 causes the accumulation of PA, enhancing multipathogen resistance (Sha et al., 2023). In our previous study, we attempted to rescue rbl1 mutant by overexpressing phosphatidate phosphohydrolase (PAH) genes. However, overexpression of PAH2 reduced the PA level but did not affect the disease resistance, which made us to reconsider the importance of PA and PAH in rice immunity. Here, we identified that mutation of PAHs caused PA accumulation and enhanced multipathogen resistance in rice and Arabidopsis.

Chloroplast to nucleus retrograde signaling is essential for cell function, acclimation to fluctuating environmental conditions, plant growth and development. The vast majority of chloroplast proteins are nuclear-encoded and must be imported into the organelle after synthesis in the cytoplasm. This import is essential for the development of fully functional chloroplasts. On the other hand, functional chloroplasts act as sensors of environmental changes and can trigger acclimatory responses that influence nuclear gene expression. Signaling via mobile transcription factors (TFs) has been recently recognized as a way of communication between organelles and the nucleus. In this study, we performed a targeted reverse genetic screen to identify novel dual-localized TFs involved in chloroplast retrograde signaling during stress responses. We found that CHLOROPLAST IMPORT APPARATUS 2 (CIA2), a TF with putative plastid transit peptide can be detected in chloroplasts and the nucleus. Further, we found that CIA2, along with its homolog CIA2-like (CIL) act in an unequally redundant manner and are involved in the regulation of Arabidopsis responses to UV-AB, high light, and heat shock. Finally, our results suggest that both CIA2 and CIL are crucial for chloroplast translation. Our results contribute to a deeper understanding of signaling events in the chloroplast-nucleus cross-talk.\n\nSignificanceWe found that a transcription factor CIA2 can be located in chloroplasts and nucleus. CIA2 and is close homolog CIL are involved in protein translation and abiotic stress responses, and we suggest that they play an essential role in retrograde signaling between these organelles.

Legumes house nitrogen-fixing endosymbiotic rhizobia in specialized polyploid cells within root nodules, which are factories of metabolic activity. We discovered that the circadian clock-associated transcriptional factor LATE ELONGATED HYPOCOTYL (LHY) affects nodulation in Medicago truncatula. By carrying out expression analysis of transcripts over time in nodules we found that the clock enables coordinated control of metabolic and regulatory processes linked to nitrogen fixation. Rhythmic transcripts in root nodules include a subset of Nodule-specific Cysteine Rich peptides (NCRs) that have the LHY-bound conserved Evening Element in their promoters. Until now, studies have suggested that NCRs act to regulate bacteroid differentiation and keep the rhizobial population in check. However, these conclusions came from the study of a few members of this very large gene family that has complex diversified spatio-temporal expression. We suggest that rhythmic expression of NCRs may be important for temporal coordination of bacterial activity with the rhythms of the plant host, in order to ensure optimal symbiosis. HighlightsO_LIThe circadian clock-associated transcriptional factor LATE ELONGATED HYPOCOTYL (LHY) impacts on successful Medicago truncatula-rhizobial symbiosis C_LIO_LIThe plant clock coordinates rhythmic patterns of metabolic and regulatory activity in nodules and drives rhythmic expression of a subset of Nodule-specific Cysteine Rich (NCR) genes. C_LIO_LIRhythmic expression of NCRs may be important for temporal coordination of bacterial activity with plant host rhythms to ensure optimal symbiosis. C_LI

Dark-germinated angiosperms develop the chloroplast precursors called etioplasts in cotyledon cells. Etioplasts develop lattice membrane structures called prolamellar bodies (PLBs), where the chlorophyll intermediate protochlorophyllide (Pchlide) forms a ternary complex with NADPH and light-dependent NADPH-Pchlide oxidoreductase (LPOR). The lipid bilayers of etioplast membranes are mainly composed of galactolipids, which play important roles in membrane-associated processes in etioplasts. Although etioplast membranes also contain two anionic lipids, phosphatidylglycerol (PG) and sulfoquinovosyldiacylglycerol (SQDG), the roles of these anionic lipids are unknown. To reveal the importance of PG and SQDG for the development of etioplasts, we characterized etiolated Arabidopsis mutants deficient in the biosynthesis of PG and SQDG. A partial deficiency in PG biosynthesis loosened the lattice structure of PLBs and impaired the insertion of Mg2+ into protoporphyrin IX, leading to a significant decrease in Pchlide content. Although a complete lack of SQDG biosynthesis did not notably affect both PLB formation and Pchlide biosynthesis, the lack of SQDG in addition to the partial deficiency of PG caused strong impairments of these processes. The results suggested that PG is required for PLB formation and Pchlide biosynthesis, whereas SQDG plays an auxiliary role in these processes. Notably, the PG deficiency and the lack of SQDG oppositely affected the dynamics of LPOR complexes after photoconversion, suggesting different involvements of PG and SQDG in the organization of LPOR complexes. Our data demonstrate pleiotropic roles of anionic lipids in etioplast development.

Rho/Rac of plant (ROP) GTPases are a plant-specific subfamily of Rho small GTP-binding proteins that function as molecular switches by being converted to the active state by guanine nucleotide exchange factors (GEFs) and to the inactive state by GTPase-activating proteins (GAPs). The bryophyte Marchantia polymorpha contains single-copy genes encoding ROP (MpROP), two types of GEFs (ROPGEF and SPIKE (SPK)), and two types of GAPs (ROPGAP and ROP enhancer (REN)). MpROP regulates the development of various organs, including the air chambers, rhizoids, and clonal propagule gemmae. While the sole PRONE-type ROPGEF, KARAPPO (MpKAR), plays an essential role in gemma initiation, little is known about the in-planta functions of other ROP regulatory factors in M. polymorpha. In this study, we focused on the functions of two types of GAPs: MpROPGAP and MpREN. Loss-of-function Mprenge single mutants showed pleiotropic defects in thallus growth, air chamber formation, rhizoid tip growth, and gemma development, whereas MpROPGAP mutants showed no detectable abnormalities. Despite the distinctive domain structures of MpROPGAP and MpREN, MpropgapgeMprenge double mutants showed more severe phenotypes than the Mprenge single mutants, suggesting redundant functions of MpROPGAP and MpREN in gametophyte organogenesis. Interestingly, overexpression of MpROPGAP, MpREN, and dominant-negative MpROP (MpROPDN) resulted in similar air chamber defects, as well as loss-of-function of MpREN and MpROPGAP and overexpression of constitutively active MpROP (MpROPCA), suggesting importance of activation/inactivation cycling (or balancing) of MpROP. Furthermore, we proved the contributions of the sole DOCK family GEF, MpSPK, to MpROP-regulated air chamber formation. In summary, our results demonstrate a significant role of the two GAPs in the development of various organs and that the two GEFs are responsible for organogenesis through the control of the MpROP active/inactive cycle in the vegetative growth of M. polymorpha.

Gene pairs resulting from whole genome duplication (WGD), so-called ohnologous genes, are retained only if at least one gene of the pair undergoes neo- or subfunctionalization. Sequence-based phylogenetic analyses of the ohnologous genes ALBOSTRIANS (HvAST/HvCMF7) and ALBOSTRIANS-LIKE (HvASL/HvCMF3) of barley (Hordeum vulgare) revealed that they belong to a newly identified subfamily of genes encoding CCT domain proteins with putative N-terminal chloroplast transit peptides. Recently, we showed that HvCMF7 is needed for chloroplast ribosome biogenesis. Here we demonstrate that mutations in HvCMF3 lead to seedlings delayed in development. They exhibit a xantha phenotype and successively develop pale green leaves. Compared to the wild type, plastids of the mutant seedlings show decreased PSII efficiency and lower amounts of ribosomal RNAs; they contain less thylakoids and grana with a higher number of more loosely stacked thylakoid membranes. Site-directed mutagenesis of HvCMF3 identified a previously unknown functional region, which is highly conserved within this subfamily of CCT domain containing proteins. HvCMF3:GFP fusion constructs localized to plastids. Hvcmf3Hvcmf7 double mutants indicated epistatic activity of HvCMF7 over HvCMF3. The chloroplast ribosome deficiency is discussed as the primary defect of the Hvcmf3 mutants. Our data suggests that HvCMF3 and HvCMF7 have similar but not identical functions.\n\nOne-sentence summaryPhylogenetic and mutant analyses of the barley protein HvCMF3 (ALBOSTRIANS-LIKE) identified, in higher plants, a subfamily of CCT domain proteins with essential function in chloroplast development.

Salicylic acid (SA) homeostasis determines also developmental senescence and is spatiotemporally controlled by various mechanisms, including biosynthesis, transport and conjugate formation. The alteration of WHIRLY1 (WHY1), a repressor of leaf natural senescence, with respect to allocation in the nucleus or chloroplast causes a perturbation in SA homeostasis, resulting in adverse plant senescence phenotypes. Loss of WHY1 resulted in a 5 days earlier SA peak compared to wild type plants which accumulated SA at 42 days after germination. SA accumulation coincided with an early leaf senescence phenotype, which could be prevented by ectopic expression of the nuclear WHY1 isoform (nWHY1). However, expressing the plastid WHY1 isoform (pWHY1) greatly enhanced cellular SA levels. A global transcriptional analysis in WHY1 loss-of-function background by expressing either pWHY1 or nWHY1 indicated that hormone metabolism related genes were most significantly altered. The pWHY1 isoform predominantly affected stress related gene expression, while the nWHY1 controlled rather developmental gene expression. Chromatin immunoprecipitation-qPCR (ChIP-qPCR) assays indicated that nWHY1 directly binds to the promoter region of isochorismate synthase (ICS1) to activate its expression at later stage, but indirectly activated S-adenosyl-L-methionine-dependent methyltransferase (BSMT1) gene expression via ethylene response factor 109 (ERF109), while repressing phenylalanine ammonia lyase (PAL1) expression via R2R3-MYB member 15 (MYB15) at the early stage of development. Interestingly, rising SA levels exerted a feedback effect by inducing nWHY1 modification and pWHY1 accumulation. Thus, the alteration of WHY1 organelle isoforms and the feedback of SA intervened in a circularly integrated regulatory network during developmental or stress-induced senescence in Arabidopsis.

Dark-induced senescence triggers significant metabolic changes that recycle resources and ensure plant survival. In this study, we identified a transcription factor OsS40-14 in rice, which can form homo-oligomers. The oss40-14 knockout mutants exhibited stay-green phenotype of primary leaf and flag leaf during dark-induced condition, with substantial retention of chlorophylls and photosynthetic capacity as well as remarkably reduced reactive oxygen species (ROS), while OsS40-14 overexpressing transgenic lines (oeOsS40-14) showed an accelerated senescence phenotype under dark-induced leaf senescence conditions. Transcriptome analysis revealed that when the detached leaves of oss40-14 and WT were treated in darkness condition for 72 hours, 1585 DEGs (|Log2FC| [&ge;]1, P value<0.05) were reprogrammed in oss40-14 relative to WT. CUT&Tag-seq analysis in protoplast transient expression of OsS40-14 system showed that OsS40-14 was 40.95% enriched in the transcription start site (TSS) of the genome. Sequence clustering analysis showed that OsS40-14 protein was mainly enriched and bound to TACCCACAAGACAC conserved elements. The seed region "ACCCA" of OsS40 proteins was identified by single nucleotide mutagenesis EMSA. The integrative analysis of transcriptome and CUT&Tag-seq datasets showed 153 OsS40-14-targeted DEGs, they mainly enriched in plastid organization and photosynthesis process at dark-induced condition in oss40-14 relative to WT. Among them, eleven candidate targets of OsS40-14 such as Glucose 6-phosphate/phosphate translocator, Na+/H+ antiporter, Catalase, Chitinase 2, Phosphate transporter 19, OsWAK32, and OsRLCK319 were directly targeted and upregulated confirmed by ChIP-PCR and RT-qPCR. It demonstrates a novel model of OsS40-14 mediating macromolecule metabolism and nutrient recycling controls the plastid organization during dark-induced leaf senescence. Significant statementInvolvement of OsS40-14 in macromolecule catabolism, nutrient recycling, and ROS homeostasis revealed a plastid organization defection of dark-induced senescence in rice

Early gene expression in arbuscular mycorrhiza (AM) and the nitrogen-fixing root nodule symbiosis (RNS) is governed by a shared regulatory complex. Yet many symbiosis-induced genes are specifically activated in only one of the two symbioses. The Lotus japonicus T-DNA insertion line T90, carrying a promoterless uidA (GUS) gene in the promoter of Calcium Binding Protein1 (CBP1) is exceptional as it exhibits GUS activity in both root endosymbioses. To identify the responsible cis- and trans-acting factors, we subjected deletion/modification series of CBP1 promoter:reporter fusions to transactivation and spatio-temporal expression analysis and screened EMS-mutagenized T90 populations for aberrant GUS expression. We identified one cis-regulatory element required for GUS expression in the epidermis and a second element, necessary and sufficient for transactivation by the Calcium and Calmodulin-dependent protein kinase (CCaMK) in combination with the transcription factor Cyclops and conferring gene expression during both AM and RNS. Lack of GUS expression in T90 white mutants could be traced to DNA hypermethylation detected in and around this element. We concluded that the CCaMK/Cyclops complex can contribute to at least three distinct gene expression patterns on its direct target promoters NIN (RNS), RAM1 (AM), and CBP1 (AM and RNS), calling for yet-to-be identified specificity-conferring factors.

O_LIPlant nucleotide-binding leucine-rich repeat receptors (NLRs) initiate immune responses and the hypersensitive response by recognizing pathogen effectors. Xa1 encodes an NLR with an N-terminal BED domain, and recognizes transcription activator-like (TAL) effectors of Xanthomonas oryzae pv. oryzae (Xoo). The molecular mechanisms controlling the recognition of TAL effectors by Xa1 and the subsequent induction of immunity remain poorly understood. C_LIO_LIXa1 interacts in the nucleus with two TAL effectors via the BED domain. We identified the AP2/ERF-type transcription factor OsERF101/OsRAP2.6 as an interactor with Xa1, and found that it also interacts with the TAL effectors. Overexpression of OsERF101 exhibited an enhanced resistance to an incompatible Xoo strain only in the presence of Xa1, indicating that OsERF101 functions as a positive regulator of Xa1-mediated immunity. Unexpectedly, oserf101 mutants also showed enhanced Xa1-dependent resistance, but in a different manner from the overexpressing plants. This result revealed an additional Xa1-mediated immune pathway that is negatively regulated by OsERF101. Furthermore, OsERF101 directly interacted with the TAL effectors. C_LIO_LIOur results show that OsERF101 regulates the recognition of TAL effectors and the Xa1-mediated activation of the immune response. These data provide new insights into the molecular mechanism of NLR-mediated immunity in plants. C_LI

The basal levels of salicylic acid (SA), an important plant hormone, vary dramatically among plant species. In the shoot, for example, the monocot plant rice contains almost 100 times higher SA levels than the dicot model plant Arabidopsis. Despite its high basal levels, neither the biosynthetic pathway nor the biological functions of SA is well understood in rice. Here, we report that the synthesis of basal SA in rice shoot is not altered in the mutant for the ISOCHORISMATE SYNTHASE (ICS) gene, but drastically reduced in the mutant for OsAIM1, which encodes a beta-oxidation enzyme in the phenylalanine ammonia-lyase (PAL) pathway. Analogous to its role in thermogenesis, compromised SA accumulation in the Osaim1 mutant led to a lower shoot temperature than wild-type plants. However, this shoot temperature defect was resulted from increased transpiration due to elevated steady-state stomatal aperture in the mutant. Furthermore, the high basal shoot SA level is required for sustained expression of WRKY45 to modulate the steady-state stomatal aperture and shoot temperature in rice. Taken together, these results provide the direct genetic evidence for the critical role of the PAL pathway in the biosynthesis of high levels of basal SA, which play an important role in the regulation of steady-state stomatal aperture to promote fitness under both normal and stress conditions.

Small molecules, peptides and miRNAs, are the crucial regulators of plant growth. Here, we show the importance of cross-talk between miPEP858a/miR858a and Phytosulfokine (PSK4) in regulating plant growth and development in Arabidopsis. Genome-wide expression analysis suggested modulated expression of PSK4 in miR858 mutant and overexpression, miR858OX, plants. The silencing of PSK4 in miR858OX plants compromised the growth, whereas over-expression of PSK4 in miR858 mutant rescued the developmental defects. The exogenous application of synthetic PSK4 further complemented the plant development in mutant plants. Exogenous treatment of synthetic miPEP858a in PSK4 mutant led to clathrin-mediated internalization of the peptide however did not enhance growth as in the case of wild-type plants. We also demonstrate that the MYB3 is an important molecular component participating in miPEP858a/miR858a-PSK4 module. Finally, our work highlights the signalling between miR858/miPEP858-MYB3-PSK4 in modulating the expression of key elements involved in auxin responses leading to the regulation of growth. One-sentence summarySignaling network between small molecules, miPEP858a/miR858a and phytosulfokine, regulates plant growth in Arabidopsis.

The extra-ribosomal functions of ribosomal proteins RPL6 and RPL23a in stress-responsiveness have emanated from our previous studies on activation tagged mutants of rice screened for water-use efficiency (Moin et al., 2016a). In the present study, we functionally validated the RPL6, a Ribosomal Protein Large subunit member for salt stress tolerance in rice. The overexpression of RPL6 resulted in tolerance to moderate (150 mM) to high (200 mM) levels of salt (NaCl) in rice. The transgenic rice plants expressing RPL6 constitutively showed better phenotypic and physiological responses with high quantum efficiency, accumulation of more chlorophyll and proline contents, and an overall increase in seed yield compared with the wild type in salt stress treatments. An iTRAQ-based comparative proteomic analysis revealed the high expression of about 333 proteins among the 4,378 DEPs in a selected overexpression line of RPL6 treated with 200 mM of NaCl. The functional analysis showed that these highly expressed proteins (HEPs) are involved in photosynthesis, ribosome and chloroplast biogenesis, ion transportation, transcription and translation regulation, phytohormone and secondary metabolite signal transduction. An in silico network analysis of HEPs predicted that RPL6 binds with translation-related proteins and helicases, which coordinately affects the activities of a comprehensive signaling network, thereby inducing tolerance and promoting growth and yield in response to salt stress. Our overall findings identified a novel candidate, RPL6 whose characterization contributed to the existing knowledge on the complexity of salt tolerance mechanism in plants.

Auxin gradients in the root cap play a crucial role in the root cap development and root tropism. In the central root cap, meristematic cells in the columella initial differentiate into gravity-sensing columella cell, and mature into border-like cells that eventually separate from the root cap. Golgi stacks exhibit distinct ultrastructural features in each cell type across the central root cap, serving as effective cell markers. This cell type-specific Golgi remodeling was inhibited in smb-3 mutant root cap. When we carried out transcriptomic analyses of isolated root cap from Col-0 and smb-3, transcript levels of numerous auxin-related genes, including those involved in auxin synthesis, transport, and signaling, were significantly altered in smb-3 mutant root caps. The auxin gradient was suppressed within the central root cap of smb-3 mutant lines and they displayed decreased sensitivity to N-1-naphthylphthalamic acid (NPA) in comparison to Col-0. Furthermore, we demonstrated that the SMB binds to the promoter regions of auxin-related genes displaying large changes in smb-3 root cap cells. Our findings suggest that SMB functions as a versatile transcription factor that regulates the expression of genes critical for local auxin distribution and responses in the root cap, thereby coordinating root cap cell differentiation and controlling root growth directions in response to external stimuli.

The beneficial element silicon (Si) plays a crucial role in mitigating salt stress in plants and can activate intracellular signal transduction pathways that reprogram the transcriptome of salt-stressed plants. However, the mechanisms by which plants perceive Si nutrition, induce the expression of Si-responsive genes, and regulate the activity of Si-responsive proteins remain inadequately understood. In this study, we found that Si enhances cell wall integrity by increasing the degree of pectin methyl-esterification and changing the cellulose microfibril arrangement and functions as a regulatory switch for the activities of cell wall integrity sensors (FER homologs OsFLR1 and OsFLR2) during rice plant adaptation to high salinity. Furthermore, the inhibition of OsFLR1 and OsFLR2 through FER-specific inhibitors could negate the beneficial effects of Si and affect the uptake and accumulation of Si in rice seedlings. In conclusion, these findings suggest that Si signaling initiation involves FER-mediated cell wall integrity signaling pathways during salt stress adaptation in rice.